Menu

Friday, April 19, 2013

The dynamics of global protein synthesis (both spatial and temporal)
is an essential parameter to characterize the cellular response under
various physiological and pathological conditions. Protein synthesis has
traditionally been monitored by metabolic labeling with 35S-methionine
that competes with natural methionine for the incorporation into newly
synthesized proteins. The detection is limited to autoradiography.

Liu et al. reported a non-radioactive alternative to analyze newly synthesized proteins in cell culture and whole organisms that is based on an alkyne analog of puromycin (Fig. 1). The cell-permeable O-Propargyl-puromycin
(Fig. 1A) incorporates into the C-terminus of translating polypeptide
chains thereby stopping translation. The resulting C-terminal alkyne
labeled proteins can be detected via Cu(I)-catalyzed click chemistry that offers the choice to introduce a Biotin group (Azides of Biotin) for subsequent purification tasks or a fluorescent group (Azides of fluorescent dyes) for subsequent microscopic imaging[1,2,3].

In contrast to previously reported non-radioactive methionine analog-approaches[5,6], methionine free-medium is not required for O-Propargyl-purmoycin-based monitoring of nascent protein synthesis.

Figure 1: O-propargyl-puromycin labels newly synthesized proteins
in cell culture and whole organisms (modified according to [1]). A) Chemical structure of O-Propargyl-puromycin (OP-puro). Visualization of incorporated OP-puro is performed via Cu(I)-catalyzed Click Chemistry (CuAAC) with Azides of fluorescent dyes or Biotin Azides.
B) Nascent protein expression in crypts of mouse small intestine was
visualized by whole-mount staining. O-Propargyl-puromycin labeled
proteins were detected with Tamra-azide (red) and nuclear DNA was
stained with OliGreen (green) (modified according to [1]) C) Newly
synthesized proteins in NIH3T3 cells are rapidly detected by incubation
with 50 µM OP-puro. OP-puro labeled proteins have been visualized by
Alexa568-azide, nuclear DNA was stained with Hoechst dye (modified
according to [1]).

Stratech Scientific
is a distributor of high quality, competitively priced, reliable
products for research laboratories throughout the UK and Europe. Please
contact us to find out which ranges we can supply in your country.